Our long term objective is to understand the molecular mechanisms by which neurotransmitters are secreted by excited nerve terminals. Our approach will be to continue our study of the biochemical mechanism of action of presynaptically acting polypeptide neurotoxins that inhibit the evoked release of neurotransmitters. In particular we will study various presynaptic neurotoxins purified from snake venoms. These toxins inhibit the evoked release of acetylcholine from motor neurons and most neurons of the autonomic nervous system. Some of these toxins are also myotoxic in that they cause muscle degeneration independent of their effects on nerve terminals. Previous work indicates that these toxins bind specifically to and decrease the conductivity of some ion channel (likely a K+ channel) that is located in nerve terminals and muscle and that is involved in Ca+ transport. We propose to identify and isolate this channel. Our experiments will be conducted with fragmented sarcoplasmic reticulum, a very simple Ca++ transport system that is also inhibited by the snake venom toxins. This inhibition is a mechanistically relevant correlate of their toxicity. The vesicle membrane components can be solubilized, purified and reconstituted into active Ca++ pumping vesicles that retain their sensitivity to the toxins. We will identify the molecular target of the toxins by two types of experiments. In the first experiment we will solubilize and reconstitute Ca++ pumping vesicles using various combinations of membrane proteins; then we will determine which protein needs to be present to allow the toxins to inhibit Ca++ uptake. In the second type of experiment we will crosslink radioactive toxin to native sarcoplasmic reticulum vesicles; then we will fractionate the vesicle proteins and identify the protein to which the toxin is crosslinked. Once the molecular target of the toxins is identified and isolated, we will insert it into planar phospholipid bilayers in order to determine whether it is an ion channel. Recent experiments indicate that at least one of the toxins directly decreases the conductivity of the sarcoplasmic reticulum K+ channel in such planar bilayers. We will examine the specificity and mechanism of this effect.